Amino acids are applied in human medicine, in the pharmaceutical industry, in the food industry and especially in animal nutrition.
Amino acids are known to be prepared by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. Due to the great importance, continuous efforts are made to improve the production processes. Said processes may be improved with respect to fermentation-related measures such as, for example, stirring and oxygen supply or the composition of the nutrient media, such as, for example, sugar concentration during the fermentation, or the working-up into product form, for example by means of ion exchange chromatography, or the intrinsic performance characteristics of the microorganism itself.
The performance characteristics of said microorganisms are improved by applying methods of mutagenesis, selection and mutant choice. This enables strains to be obtained which are resistant to antimetabolites or auxotrophic for metabolites which are of regulatory importance, and produce amino acids. A known antimetabolite is the lysine analog S-(2-aminoethyl)-L-cysteine (AEC).
For some years now, methods of recombinant DNA technology have likewise been employed in order to improve L-amino acid-producing Corynebacterium strains, by amplifying individual amino acid biosynthesis genes and studying the effect on amino acid production. A summary on a wide variety of aspects of the genetics, the metabolism and the biotechnology of Corynebacterium glutamicum can be found in Pühler (chief ed.) in Journal of Biotechnology 104 (1-3), 1-338, 2003.
The nucleotide sequence of the gene coding for glucose 6-phosphate dehydrogenase of Corynebacterium glutamicum is generally accessible, inter alia in the database of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, Md., USA). It can furthermore be found as sequence no. 243 (=AX065117) in the patent application WO 01/00844.
WO 01/70995 describes an improvement in the fermentative production of L-amino acids by coryneform bacteria due to amplification of the zwf gene.
WO 01/98472, WO 03/042389 and US 2003/0175911 A1 report novel mutations in the zwf gene.
Moritz et al. (European Journal of Biochemistry 267, 3442-3452 (2000)) report physiological and biochemical studies on glucose 6-phosphate dehydrogenase of Corynebacterium glutamicum. According to studies by Moritz et al., glucose 6-phosphate dehydrogenase consists of a Zwf subunit and an OpcA subunit.
The microbial biosynthesis of L-amino acids in coryneform bacteria is a complex system and linked on multiple levels to various other metabolic pathways in the cell. It is therefore not possible to predict which mutation alters the catalytic activity of glucose 6-phosphate dehydrogenase in such a way that production of L-amino acids is improved. It is therefore desirable to have available further variants of glucose 6-phosphate dehydrogenase.
For reasons of better clarity, SEQ ID NO:1 depicts the nucleotide sequence of the zwf gene coding for glucose 6-phosphate dehydrogenase and, respectively, of the zwf gene coding for the Zwf subunit of glucose 6-phosphate dehydrogenase from Corynebacterium glutamicum (“wild type gene”), according to the information of the NCBI database, and SEQ ID NO:2 and 4 depict the amino acid sequence derived therefrom of the encoded glucose 6-phosphate dehydrogenase. In addition, SEQ ID NO:3 indicates nucleotide sequences located upstream and downstream.